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14 results on '"Marie Hélène Le Du"'

1. BRCA2 binding through a cryptic repeated motif to HSF2BP oligomers does not impact meiotic recombination

Author

Marie-Hélène Le Du, Maarten W. Paul, Roland Kanaar, Esther Sleddens-Linkels, Rania Ghouil, Alberto M. Pendás, Pierre Legrand, Lieke Koornneef, Yvette van Loon, Natalia Felipe-Medina, Jasper Veerman, Alex N. Zelensky, Jeroen Essers, Willy M. Baarends, Sari E. van Rossum-Fikkert, Sophie Zinn-Justin, Alex Maas, Simona Miron, Ministerio de Economía y Competitividad (España), Instituto de Salud Carlos III, Junta de Castilla y León, Asociación Española Contra el Cáncer, Fundación CRIS contra el Cáncer, European Commission, Developmental Biology, Molecular Genetics, Radiation Oncology, Cell biology, and Surgery

Subjects
Male, Magnetic Resonance Spectroscopy, endocrine system diseases, DNA recombination, Science, RAD51, General Physics and Astronomy, Reproductive biology, Cell Cycle Proteins, Crystallography, X-Ray, General Biochemistry, Genetics and Molecular Biology, Article, 03 medical and health sciences, Exon, Mice, 0302 clinical medicine, Meiosis, biology.animal, Recombinase, Animals, Humans, Protein Interaction Domains and Motifs, skin and connective tissue diseases, Tumour-suppressor proteins, Homologous Recombination, Spermatogenesis, neoplasms, Cells, Cultured, 030304 developmental biology, X-ray crystallography, Sequence Deletion, Phenocopy, BRCA2 Protein, 0303 health sciences, Multidisciplinary, biology, Chemistry, General Chemistry, female genital diseases and pregnancy complications, Cell biology, Armadillo, Models, Animal, DMC1, Female, Homologous recombination, 030217 neurology & neurosurgery
Abstract

© The Author(s) 2021., BRCA2 and its interactors are required for meiotic homologous recombination (HR) and fertility. Loss of HSF2BP, a BRCA2 interactor, disrupts HR during spermatogenesis. We test the model postulating that HSF2BP localizes BRCA2 to meiotic HR sites, by solving the crystal structure of the BRCA2 fragment in complex with dimeric armadillo domain (ARM) of HSF2BP and disrupting this interaction in a mouse model. This reveals a repeated 23 amino acid motif in BRCA2, each binding the same conserved surface of one ARM domain. In the complex, two BRCA2 fragments hold together two ARM dimers, through a large interface responsible for the nanomolar affinity — the strongest interaction involving BRCA2 measured so far. Deleting exon 12, encoding the first repeat, from mBrca2 disrupts BRCA2 binding to HSF2BP, but does not phenocopy HSF2BP loss. Thus, results herein suggest that the high-affinity oligomerization-inducing BRCA2-HSF2BP interaction is not required for RAD51 and DMC1 recombinase localization in meiotic HR., AP: Ministry of Economy and Competitiveness of Spain (BFU2015–71371-R), the Instituto de Salud Carlos III through CIBERONC, Junta de Castilla y León (CSI146P20), the Scientific Foundation of the Spanish Association Against Cancer (AECC), ALMOM, ACMUMA and the CRIS Cancer Foundation. JCM is funded by the Instituto de Salud Carlos III through a Miguel Servet program (CP12/03073 and CPII17/00015) and receives research support from the same institution (PI18/00796). LGS is recipient of a predoctoral contract (BES-2016-077748). IRP is recipient of a predoctoral contract (CSI030–18). SGA is recipient of a predoctoral contract from the MINECO (BES-2013-065223). Work carried out in our laboratory receives support from the European Community through the Regional Development Funding Program (FEDER).

Published
2021
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2. Molecular basis of the dual role of the Mlh1-Mlh3 endonuclease in MMR and in meiotic crossover formation

Author

Lepakshi Ranjha, Pierre Chervy, Valérie Borde, Marie-Hélène Le Du, Laurent Maloisel, Jean-Baptiste Charbonnier, Giordano Reginato, Aurélien Thureau, Virginie Ropars, Emmanuelle Martini, Pierre Legrand, Jessica Andreani, Aurore Sanchez, Céline Adam, Petr Cejka, Jingqi Dai, Raphael Guerois, Carine Tellier-Lebegue, Institut de Biologie Intégrative de la Cellule (I2BC), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)

Subjects
Models, Molecular, congenital, hereditary, and neonatal diseases and abnormalities, Saccharomyces cerevisiae Proteins, DNA Repair, [SDV]Life Sciences [q-bio], Saccharomyces cerevisiae, Context (language use), MLH3, MLH1, environment and public health, DNA Mismatch Repair, 03 medical and health sciences, Holliday junction, 030304 developmental biology, 0303 health sciences, Multidisciplinary, Binding Sites, biology, Chemistry, 030302 biochemistry & molecular biology, fungi, Recombinational DNA Repair, Biological Sciences, biology.organism_classification, Endonucleases, Cell biology, DNA-Binding Proteins, enzymes and coenzymes (carbohydrates), Meiosis, MutL Proteins, DNA mismatch repair, CTD, Homologous recombination, MutL Protein Homolog 1
Abstract

International audience; In budding yeast, the MutL homolog heterodimer Mlh1-Mlh3 (MutLγ) plays a central role in the formation of meiotic crossovers. It is also involved in the repair of a subset of mismatches besides the main mismatch repair (MMR) endonuclease Mlh1-Pms1 (MutLα). The heterodimer interface and endonuclease sites of MutLγ and MutLα are located in their C-terminal domain (CTD). The molecular basis of MutLγ’s dual roles in MMR and meiosis is not known. To better understand the specificity of MutLγ, we characterized the crystal structure of Saccharomyces cerevisiae MutLγ(CTD). Although MutLγ(CTD) presents overall similarities with MutLα(CTD), it harbors some rearrangement of the surface surrounding the active site, which indicates altered substrate preference. The last amino acids of Mlh1 participate in the Mlh3 endonuclease site as previously reported for Pms1. We characterized mlh1 alleles and showed a critical role of this Mlh1 extreme C terminus both in MMR and in meiotic recombination. We showed that the MutLγ(CTD) preferentially binds Holliday junctions, contrary to MutLα(CTD). We characterized Mlh3 positions on the N-terminal domain (NTD) and CTD that could contribute to the positioning of the NTD close to the CTD in the context of the full-length MutLγ. Finally, crystal packing revealed an assembly of MutLγ(CTD) molecules in filament structures. Mutation at the corresponding interfaces reduced crossover formation, suggesting that these superstructures may contribute to the oligomer formation proposed for MutLγ. This study defines clear divergent features between the MutL homologs and identifies, at the molecular level, their specialization toward MMR or meiotic recombination functions.

Published
2021
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3. Di-phosphorylated BAF shows altered structural dynamics and binding to DNA, but interacts with its nuclear envelope partners

Author

Virginie Ropars, Guillaume Hoffmann, Sophie Zinn-Justin, Marie-Hélène Le Du, Philippe Cuniasse, Ambre Petitalot, Robert Thai, Camille Samson, Agathe Marcelot, Stevens Dubois, José A. Márquez, Simona Miron, François-Xavier Theillet, Département Plateforme (PF I2BC), Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Service d'Ingénierie Moléculaire pour la Santé (ex SIMOPRO) (SIMoS), Médicaments et Technologies pour la Santé (MTS), Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), High Throughput Crystallization Laboratory, European Molecular Biology Laboratory Grenoble Outstation, European Molecular Biology Laboratory, ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), European Project: 653706,H2020,H2020-INFRAIA-2014-2015,iNEXT(2015), European Project: 871037,iNEXT-Discovery (H2020-EU.1.4.1.2), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), and European Molecular Biology Laboratory [Grenoble] (EMBL)

Subjects
AcademicSubjects/SCI00010, Emerin, Protein Serine-Threonine Kinases, Biology, Protein Structure, Secondary, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, Genetics, Humans, Amino Acid Sequence, Phosphorylation, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Ternary complex, Gene, 030304 developmental biology, 0303 health sciences, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Kinase, Gene regulation, Chromatin and Epigenetics, Intracellular Signaling Peptides and Proteins, Membrane Proteins, Nuclear Proteins, DNA, Cell cycle, Lamin Type A, 3. Good health, Cell biology, DNA-Binding Proteins, chemistry, ddc:540, Protein Processing, Post-Translational, 030217 neurology & neurosurgery, Lamin, Protein Binding
Abstract

Barrier-to-autointegration factor (BAF), encoded by the BANF1 gene, is an abundant and ubiquitously expressed metazoan protein that has multiple functions during the cell cycle. Through its ability to cross-bridge two double-stranded DNA (dsDNA), it favours chromosome compaction, participates in post-mitotic nuclear envelope reassembly and is essential for the repair of large nuclear ruptures. BAF forms a ternary complex with the nuclear envelope proteins lamin A/C and emerin, and its interaction with lamin A/C is defective in patients with recessive accelerated aging syndromes. Phosphorylation of BAF by the vaccinia-related kinase 1 (VRK1) is a key regulator of BAF localization and function. Here, we demonstrate that VRK1 successively phosphorylates BAF on Ser4 and Thr3. The crystal structures of BAF before and after phosphorylation are extremely similar. However, in solution, the extensive flexibility of the N-terminal helix α1 and loop α1α2 in BAF is strongly reduced in di-phosphorylated BAF, due to interactions between the phosphorylated residues and the positively charged C-terminal helix α6. These regions are involved in DNA and lamin A/C binding. Consistently, phosphorylation causes a 5000-fold loss of affinity for dsDNA. However, it does not impair binding to lamin A/C Igfold domain and emerin nucleoplasmic region, which leaves open the question of the regulation of these interactions.

Published
2021
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4. Structural analysis of the ternary complex between lamin A/C, BAF and emerin identifies an interface disrupted in autosomal recessive progeroid diseases

Author

Ana-Andreea Arteni, Virginie Ropars, Marie-Hélène Le Du, Ambre Petitalot, Florian Celli, Camille Samson, Naïma Nhiri, Eric Jacquet, Sophie Zinn-Justin, Isaline Herrada, Brigitte Buendia, Enveloppe Nucléaire, Télomères et Réparation de l’ADN (INTGEN), Département Biochimie, Biophysique et Biologie Structurale (B3S), Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Institut de Chimie des Substances Naturelles (ICSN), Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), Unité de Biologie Fonctionnelle et Adaptative (BFA (UMR_8251 / U1133)), Université Paris Diderot - Paris 7 (UPD7)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS), Cryo-microscopie électronique (CRYOEM), Département Plateforme (PF I2BC), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Saclay, Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Saclay-Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Saclay-Institut de Biologie Intégrative de la Cellule (I2BC), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Saclay-Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Saclay, Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), and ANR-10-INBS-05-01/10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010)

Subjects
0301 basic medicine, Models, Molecular, Nuclear Envelope, [SDV]Life Sciences [q-bio], Protein domain, Emerin, Genes, Recessive, Plasma protein binding, Biology, medicine.disease_cause, Crystallography, X-Ray, Progeroid syndromes, 03 medical and health sciences, Progeria, Protein Domains, Structural Biology, Genetics, medicine, Humans, Gene, Mutation, Membrane Proteins, Nuclear Proteins, medicine.disease, Lamin Type A, Chromatin, Cell biology, DNA-Binding Proteins, 030104 developmental biology, Multiprotein Complexes, Protein Multimerization, Lamin, Protein Binding
Abstract

International audience; Lamins are the main components of the nucleoskeleton. Whereas their 3D organization was recently described using cryoelectron tomography, no structural data highlights how they interact with their partners at the interface between the inner nuclear envelope and chromatin. A large number of mutations causing rare genetic disorders called laminopathies were identified in the C-terminal globular Igfold domain of lamins A and C. We here present a first structural description of the interaction between the lamin A/C immunoglobulin-like domain and emerin, a nuclear envelope protein. We reveal that this lamin A/C domain both directly binds self-assembled emerin and interacts with monomeric emerin LEM domain through the dimeric chromatin-associated Barrier-to-Autointegration Factor (BAF) protein. Mutations causing autosomal recessive progeroid syndromes specifically impair proper binding of lamin A/C domain to BAF, thus destabilizing the link between lamin A/C and BAF in cells. Recent data revealed that, during nuclear assembly, BAF's ability to bridge distant DNA sites is essential for guiding membranes to form a single nucleus around the mitotic chromosome ensemble. Our results suggest that BAF interaction with lamin A/C also plays an essential role, and that mutations associated with progeroid syndromes leads to a dysregulation of BAF-mediated chromatin organization and gene expression.

Published
2018
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5. Substrate and Reaction Specificity of Mycobacterium tuberculosis Cytochrome P450 CYP121

Author

Steven Dubois, Guillaume Grach, Alain Lecoq, Robert Thai, Marie-Hélène Le Du, Matthieu Fonvielle, Muriel Gondry, Pascal Belin, Mickaël Jacquet, and Olivier Lequin

Subjects
chemistry.chemical_classification, 0303 health sciences, Chemistry, Stereochemistry, Aryl, Substrate (chemistry), Cell Biology, Nuclear magnetic resonance spectroscopy, Plasma protein binding, 010402 general chemistry, Ligand (biochemistry), 01 natural sciences, Biochemistry, Enzyme structure, 3. Good health, 0104 chemical sciences, 03 medical and health sciences, chemistry.chemical_compound, Oxidoreductase, Molecular Biology, Binding selectivity, 030304 developmental biology
Abstract

Cytochrome P450 CYP121 is essential for the viability of Mycobacterium tuberculosis. Studies in vitro show that it can use the cyclodipeptide cyclo(l-Tyr-l-Tyr) (cYY) as a substrate. We report an investigation of the substrate and reaction specificities of CYP121 involving analysis of the interaction between CYP121 and 14 cYY analogues with various modifications of the side chains or the diketopiperazine (DKP) ring. Spectral titration experiments show that CYP121 significantly bound only cyclodipeptides with a conserved DKP ring carrying two aryl side chains in l-configuration. CYP121 did not efficiently or selectively transform any of the cYY analogues tested, indicating a high specificity for cYY. The molecular determinants of this specificity were inferred from both crystal structures of CYP121-analog complexes solved at high resolution and solution NMR spectroscopy of the analogues. Bound cYY or its analogues all displayed a similar set of contacts with CYP121 residues Asn85, Phe168, and Trp182. The propensity of the cYY tyrosyl to point toward Arg386 was dependent on the presence of the DKP ring that limits the conformational freedom of the ligand. The correct positioning of the hydroxyl of this tyrosyl was essential for conversion of cYY. Thus, the specificity of CYP121 results from both a restricted binding specificity and a fine-tuned P450 substrate relationship. These results document the catalytic mechanism of CYP121 and improve our understanding of its function in vivo. This work contributes to progress toward the design of inhibitors of this essential protein of M. tuberculosis that could be used for antituberculosis therapy.

Published
2013
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6. TRF2-Mediated Control of Telomere DNA Topology as a Mechanism for Chromosome-End Protection

Author

Marie-Hélène Le Du, Simona Miron, Bei Pei, Hong Wang, Eric Gilson, Parminder Kaur, Arturo Londoño-Vallejo, Jing Ye, Emilie Jaune, Aaron Mendez-Bermudez, Sabrina Pisano, Marie-Josèphe Giraud-Panis, Vincent Fraisier, Julien Cherfils-Vicini, Nadir Djerbi, Eric Aeby, Delphine Benarroch-Popivker, Serge Bauwens, Kevin Foucher, Chrysa M Latrick, Liudmyla Lototska, Institut de Recherche sur le Cancer et le Vieillissement (IRCAN), Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015 - 2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015 - 2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA), International Laboratory in Hematology and Cancer, Shanghai Jiao Tong University School of Medicine, Physics Department, North Carolina State University [Raleigh] (NC State), University of North Carolina System (UNC)-University of North Carolina System (UNC), Compartimentation et dynamique cellulaires (CDC), Centre National de la Recherche Scientifique (CNRS)-Institut Curie [Paris]-Université Pierre et Marie Curie - Paris 6 (UPMC), Swiss Institute for Experimental Cancer Research - Lausanne (ISREC), Swiss Institute for Experimental Cancer Research, Enveloppe Nucléaire, Télomères et Réparation de l’ADN (INTGEN), Département Biochimie, Biophysique et Biologie Structurale (B3S), Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Dynamique de l'information génétique : bases fondamentales et cancer (DIG CANCER), Dpt génétique médicale [CHU Nice], Centre Hospitalier Universitaire de Nice (CHU Nice), ANR-10-BLAN-1512,TELOLOOP,Rôle de la protéine TRF2 et de ses partenaires dans la formation et la stabilité de la t-loop des télomères humains(2010), ANR-11-LABX-0028,SIGNALIFE,Réseau d'Innovation sur les Voies de Signalisation en Sciences de la Vie(2011), Institut de Recherche sur le Cancer et le Vieillissement ( IRCAN ), Université Nice Sophia Antipolis ( UNS ), Université Côte d'Azur ( UCA ) -Université Côte d'Azur ( UCA ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Centre National de la Recherche Scientifique ( CNRS ), North Carolina State University [Raleigh] ( NCSU ), Compartimentation et dynamique cellulaires ( CDC ), Université Pierre et Marie Curie - Paris 6 ( UPMC ) -INSTITUT CURIE-Centre National de la Recherche Scientifique ( CNRS ), Swiss Institute for Experimental Cancer Research - Lausanne ( ISREC ), Enveloppe Nucléaire, Télomères et Réparation de l’ADN ( INTGEN ), Département Biochimie, Biophysique et Biologie Structurale ( B3S ), Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ), Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ), Dynamique de l'information génétique : bases fondamentales et cancer ( DIG CANCER ), CHU Nice, ANR-1582-30020690,TELOLOOP,ANR-1582-30020690, ANR : LABEX SIGNALIFE,ANR-11-LABX-0028-01, Université Nice Sophia Antipolis (1965 - 2019) (UNS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut Curie [Paris]-Centre National de la Recherche Scientifique (CNRS), COMUE Université Côte d'Azur (2015 - 2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015 - 2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Telomeres and Cancer Laboratory, Institut Curie [Paris], School of Mechanical Engineering, Sungkyunkwan University [Suwon] (SKKU), Laboratoire de Biologie Moléculaire de la Cellule (LBMC), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Université de Lyon

Subjects
Models, Molecular, 0301 basic medicine, DNA End-Joining Repair, [SDV]Life Sciences [q-bio], Ataxia Telangiectasia Mutated Proteins, TRF2, [SDV.BC.BC]Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], medicine.disease_cause, Shelterin Complex, chemistry.chemical_compound, Telomeric Repeat Binding Protein 2, Base Pairing, ComputingMilieux_MISCELLANEOUS, Telomere-binding protein, Mutation, telomere, RAP1, DNA topology, DNA wrapping, Signal Transduction, DNA damage, Base pair, Telomere-Binding Proteins, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Biology, Topology, Article, 03 medical and health sciences, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], medicine, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Molecular Biology, [ SDV ] Life Sciences [q-bio], Lysine, DNA, Cell Biology, Shelterin, Molecular biology, Protein Structure, Tertiary, Telomere, enzymes and coenzymes (carbohydrates), 030104 developmental biology, chemistry, Nucleic Acid Conformation, DNA Damage, HeLa Cells
Abstract

International audience; The shelterin proteins protect telomeres against activation of the DNA damage checkpoints and recombinational repair. We show here that a dimer of the shelterin subunit TRF2 wraps ∼ 90 bp of DNA through several lysine and arginine residues localized around its homodimerization domain. The expression of a wrapping-deficient TRF2 mutant, named Top-less, alters telomeric DNA topology, decreases the number of terminal loops (t-loops), and triggers the ATM checkpoint, while still protecting telomeres against non-homologous end joining (NHEJ). In Top-less cells, the protection against NHEJ is alleviated if the expression of the TRF2-interacting protein RAP1 is reduced. We conclude that a distinctive topological state of telomeric DNA, controlled by the TRF2-dependent DNA wrapping and linked to t-loop formation, inhibits both ATM activation and NHEJ. The presence of RAP1 at telomeres appears as a backup mechanism to prevent NHEJ when topology-mediated telomere protection is impaired.

Published
2016
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7. The orientation of the C-terminal domain of the Saccharomyces cerevisiae Rap1 protein is determined by its binding to DNA

Author

Rachel Lescasse, Patrick Weber, Javier Pérez, Marie-Hélène Le Du, Simona Miron, Bertrand Raynal, Sylvaine Gasparini, Gabriel David, B. Matot, Sophie Zinn-Justin, Bertrand Castaing, Yann-Vaï Le Bihan, Protéines membranaires transductrices d'énergie (PMTE), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Service d'Instabilité Génétique Réparation Recombinaison (SIGRR), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Synchrotron SOLEIL (SSOLEIL), Centre National de la Recherche Scientifique (CNRS), Centre de biophysique moléculaire (CBM), Université d'Orléans (UO)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Cristallogenèse et Diffraction des Rayons X (Plate-forme/PF6), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Biophysique des macromolécules et de leurs interactions (Plate-forme), Institut Pasteur [Paris] (IP), Commissariat à l'Energie Atomique, Centre National de la Recherche Scientifique, French ‘Agence National pour la Recherche’ (ANR-06-BLAN-0076). Funding for open access charge: Commissariat à l'Energie Atomique., ANR-06-BLAN-0076,NHEJ&TELO,Double-strand break repair and genome stability: the Non-Homologous End Joining pathway and its suppression at telomeres(2006), Université d'Orléans (UO)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), Laboratoire de Physique des Solides (LPS), Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), Chemistry, Food Chemsitry, University of Hamburg, and Laboratoire de Biologie Structurale et Radiobiologie (LBSR)

Subjects
Models, Molecular, BUDDING YEAST, Crystallography, X-Ray, Shelterin Complex, MESH: Telomere-Binding Proteins, MESH: Protein Structure, Tertiary, chemistry.chemical_compound, MESH: Saccharomyces cerevisiae Proteins, 0302 clinical medicine, X-Ray Diffraction, Transcription (biology), Structural Biology, ANGLE SCATTERING DATA, MESH: Nuclear Magnetic Resonance, Biomolecular, Silent Information Regulator Proteins, Saccharomyces cerevisiae, Genetics, Telomere-binding protein, 0303 health sciences, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], biology, YEAST RAP1, MESH: DNA, MESH: X-Ray Diffraction, ASSOCIATION, MESH: Transcription Factors, SEPARATION, REPRESSOR ACTIVATOR PROTEIN-1, MESH: Models, Molecular, Binding domain, endocrine system, Saccharomyces cerevisiae Proteins, HMG-box, Saccharomyces cerevisiae, Telomere-Binding Proteins, 03 medical and health sciences, Scattering, Small Angle, TARGET SITES, MESH: Silent Information Regulator Proteins, Saccharomyces cerevisiae, Nuclear Magnetic Resonance, Biomolecular, MESH: Scattering, Small Angle, 030304 developmental biology, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, DNA-binding domain, DNA, MESH: Crystallography, X-Ray, biology.organism_classification, Protein Structure, Tertiary, DNA binding site, BIOLOGICAL MACROMOLECULES, enzymes and coenzymes (carbohydrates), chemistry, TELOMERE LENGTH, Biophysics, TRANSCRIPTIONAL ACTIVATION, 030217 neurology & neurosurgery, Transcription Factors
Abstract

International audience; Rap1 is an essential DNA-binding factor from the yeast Saccharomyces cerevisiae involved in transcription and telomere maintenance. Its binding to DNA targets Rap1 at particular loci, and may optimize its ability to form functional macromolecular assemblies. It is a modular protein, rich in large potentially unfolded regions, and comprising BRCT, Myb and RCT well-structured domains. Here, we present the architectures of Rap1 and a Rap1/DNA complex, built through a step-by-step integration of small angle X-ray scattering, X-ray crystallography and nuclear magnetic resonance data. Our results reveal Rap1 structural adjustment upon DNA binding that involves a specific orientation of the C-terminal (RCT) domain with regard to the DNA binding domain (DBD). Crystal structure of DBD in complex with a long DNA identifies an essential wrapping loop, which constrains the orientation of the RCT and affects Rap1 affinity to DNA. Based on our structural information, we propose a model for Rap1 assembly at telomere.

Published
2011
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8. Structure of the MutL$\alpha$ C-terminal domain reveals how Mlh1 contributes to Pms1 endonuclease site

Author

Bernard Gilquin, Floriana Londino, Josan A Márquez, Pierre Bonnesoeur, Claudine Dherin, Serge Boiteux, Marie-Hélène Le Du, Carine Tellier-Lebegue, Muriel Gondry, Jean-Baptiste Charbonnier, Pierre Legrand, Mireille Moutiez, Cathy Quemener, Emeric Gueneau, Laboratoire de Biologie Structurale et Radiobiologie (LBSR), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Radiobiologie moléculaire et cellulaire (RMC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Synchrotron SOLEIL (SSOLEIL), Centre National de la Recherche Scientifique (CNRS), Equipe de Chimie Organique et Biologie Structurale (ECOBS), Institut national des sciences appliquées Rouen Normandie (INSA Rouen Normandie), Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Normandie Université (NU), Systèmes membranaires, photobiologie, stress et détoxification (SMPSD - UMR 8221), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Institut de Biologie et de Technologies de Saclay (IBITECS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay, Centre de biophysique moléculaire (CBM), Université d'Orléans (UO)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), European Project: 227764,EC:FP7:INFRA,FP7-INFRASTRUCTURES-2008-1,PCUBE(2009), Université d'Orléans (UO)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), and Université Paris-Saclay-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)

Subjects
Models, Molecular, congenital, hereditary, and neonatal diseases and abnormalities, HMG-box, Saccharomyces cerevisiae, MLH1, Protein Structure, Secondary, 03 medical and health sciences, Endonuclease, 0302 clinical medicine, Protein structure, MutL Proteins, Structural Biology, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Molecular Biology, neoplasms, Adaptor Proteins, Signal Transducing, 030304 developmental biology, Genetics, 0303 health sciences, biology, Chemistry, Nuclear Proteins, nutritional and metabolic diseases, Endonucleases, biology.organism_classification, digestive system diseases, DNA Repair Enzymes, 030220 oncology & carcinogenesis, biology.protein, MutL Protein Homolog 1, Homologous recombination
Abstract

International audience; Mismatch-repair factors have a prominent role in surveying eukaryotic DNA-replication fidelity and in ensuring correct meiotic recombination. These functions depend on MutL-homolog heterodimers with Mlh1. In humans, MLH1 mutations underlie half of hereditary nonpolyposis colorectal cancers (HNPCCs). Here we report crystal structures of the MutLα (Mlh1-Pms1 heterodimer) C-terminal domain (CTD) from $Saccharomyces\ cerevisiae$, alone and in complex with fragments derived from Mlh1 partners. These structures reveal structural rearrangements and additional domains in MutLα as compared to the bacterial MutL counterparts and show that the strictly conserved C terminus of Mlh1 forms part of the Pms1 endonuclease site. The structures of the ternary complexes between MutLα(CTD) and Exo1 or Ntg2 fragments reveal the binding mode of the MIP-box motif shared by several Mlh1 partners. Finally, the structures provide a rationale for the deleterious impact of MLH1 mutations in HNPCCs.

Published
2013
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9. Effect of Rap1 binding on DNA distortion and potassium permanganate hypersensitivity

Author

Eric Gilson, Marie-Josèphe Giraud-Panis, Simona Miron, Eric Le Cam, Olivier Piétrement, Bianca Sclavi, B. Matot, Yann-Vaï Le Bihan, Marie-Hélène Le Du, Sylvaine Gasparini, Centre de biophysique moléculaire (CBM), Université d'Orléans (UO)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Institut de Myologie, Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Université de Reims Champagne-Ardenne (URCA), Signalisation, noyaux et innovations en cancérologie (UMR8126), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), Institut Gustave Roussy (IGR), Laboratoire de Biologie Moléculaire de la Cellule (LBMC), École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Biologie Structurale et Radiobiologie (LBSR), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institut de Recherche sur le Cancer et le Vieillissement (IRCAN), Université Nice Sophia Antipolis (1965 - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA), Laboratoire de biologie et pharmacologie appliquée (LBPA), Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Ecole Normale Supérieure Paris-Saclay (ENS Paris Saclay), Imagerie intégrative de la molécule à l'organisme, Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Service de Bioénergétique, Biologie Stucturale, et Mécanismes (SB2SM), Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Le Cam, Eric, Université Nice Sophia Antipolis (... - 2019) (UNS), Université d'Orléans (UO)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), Centre National de la Recherche Scientifique (CNRS)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Association française contre les myopathies (AFM-Téléthon)-Sorbonne Université (SU), Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), and École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL)

Subjects
endocrine system, Saccharomyces cerevisiae Proteins, [SDV]Life Sciences [q-bio], Telomere-Binding Proteins, Mutant, Saccharomyces cerevisiae, Arginine, Crystallography, X-Ray, Shelterin Complex, Cytosine, 03 medical and health sciences, chemistry.chemical_compound, Nucleic acid thermodynamics, 0302 clinical medicine, Potassium Permanganate, Structural Biology, Binding site, DNA, Fungal, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, 0303 health sciences, biology, Permanganate, Hydrogen Bonding, General Medicine, biology.organism_classification, Solutions, [SDV] Life Sciences [q-bio], enzymes and coenzymes (carbohydrates), Potassium permanganate, chemistry, Biochemistry, Biophysics, Nucleic Acid Conformation, 030217 neurology & neurosurgery, DNA, Protein Binding, Transcription Factors
Abstract

Repressor activator protein 1 (Rap1) is an essential factor involved in transcription and telomere stability in the budding yeast Saccharomyces cerevisiae. Its interaction with DNA causes hypersensitivity to potassium permanganate, suggesting local DNA melting and/or distortion. In this study, various Rap1-DNA crystal forms were obtained using specifically designed crystal screens. Analysis of the DNA conformation showed that its distortion was not sufficient to explain the permanganate reactivity. However, anomalous data collected at the Mn edge using a Rap1-DNA crystal soaked in potassium permanganate solution indicated that the DNA conformation in the crystal was compatible with interaction with permanganate ions. Sequence-conservation analysis revealed that double-Myb-containing Rap1 proteins all carry a fully conserved Arg580 at a position that may favour interaction with permanganate ions, although it is not involved in the hypersensitive cytosine distortion. Permanganate reactivity assays with wild-type Rap1 and the Rap1[R580A] mutant demonstrated that Arg580 is essential for hypersensitivity. AFM experiments showed that wild-type Rap1 and the Rap1[R580A] mutant interact with DNA over 16 successive binding sites, leading to local DNA stiffening but not to accumulation of the observed local distortion. Therefore, Rap1 may cause permanganate hypersensitivity of DNA by forming a pocket between the reactive cytosine and Arg580, driving the permanganate ion towards the C5-C6 bond of the cytosine.

Published
2013
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10. One Identity or More for Telomeres?

Author

Sabrina Pisano, Marie-Hélène Le Du, Eric Gilson, Bei Pei, Marie-Josèphe Giraud-Panis, Delphine Benarroch-Popivker, Institut de Recherche sur le Cancer et le Vieillissement (IRCAN), Université Nice Sophia Antipolis (... - 2019) (UNS), and COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA)

Subjects
Cancer Research, [SDV]Life Sciences [q-bio], capping complexes, Review Article, Computational biology, Biology, lcsh:RC254-282, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, Transcription (biology), chromosome, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, Genetics, 0303 health sciences, Telomere biology, telomeric chromatin organization, RNA, Robustness (evolution), Cell cycle, lcsh:Neoplasms. Tumors. Oncology. Including cancer and carcinogens, telomeres, Telomere, Nucleoprotein, DNA topology, chemistry, Oncology, 030217 neurology & neurosurgery, DNA
Abstract

A major issue in telomere research is to understand how the integrity of chromosome ends is controlled. The fact that different types of nucleoprotein complexes have been described at the telomeres of different organisms raises the question of whether they have in common a structural identity that explains their role in chromosome protection. We will review here how telomeric nucleoprotein complexes are structured, comparing different organisms and trying to link these structures to telomere biology. It emerges that telomeres are formed by a complex and specific network of interactions between DNA, RNA, and proteins. The fact that these interactions and associated activities are reinforcing each other might help to guarantee the robustness of telomeric functions across the cell cycle and in the event of cellular perturbations. We will also discuss the recent notion that telomeres have evolved specific systems to overcome the DNA topological stress generated during their replication and transcription. This will lead to revisit the way we envisage the functioning of telomeric complexes since the regulation of topology is central to DNA stability, replication, recombination, and transcription as well as to chromosome higher-order organization.

Published
2013
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11. structural identity of telomeric complexes

Author

Marie-Hélène Le Du, Anaïs Poulet, Marie-Josèphe Giraud-Panis, Eric Gilson, Sabrina Pisano, Système membranaires, photobiologie, stress et détoxication (SMPSD), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Institut de Recherche sur le Cancer et le Vieillissement (IRCAN), Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA), Université Nice Sophia Antipolis (1965 - 2019) (UNS), Laboratoire Joliot Curie, École normale supérieure de Lyon (ENS de Lyon)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de biologie et pathologie des génomes, Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratoire de Biologie Moléculaire de la Cellule (LBMC), École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
DNA Repair, DNA repair, [SDV]Life Sciences [q-bio], Biophysics, Saccharomyces cerevisiae, Computational biology, Cell fate determination, Biology, Biochemistry, Shelterin, 03 medical and health sciences, chemistry.chemical_compound, Structural Biology, Schizosaccharomyces, Genetics, Animals, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, DNA binding, Molecular Biology, ComputingMilieux_MISCELLANEOUS, CST, 030304 developmental biology, Mammals, Telomere-binding protein, 0303 health sciences, Oxytricha, 030302 biochemistry & molecular biology, Genetic Variation, Proteins, Robustness (evolution), [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, DNA, Cell Biology, Telomere, Nucleoprotein, chemistry, Tandem Repeat Sequences, RNA
Abstract

International audience; A major issue in telomere research is to understand how the integrity of chromosome ends is controlled. Although several nucleoprotein complexes have been described at the telomeres of different organisms, it is still unclear how they confer a structural identity to chromosome ends in order to mask them from DNA repair and to ensure their proper replication. In this review, we describe how telomeric nucleoprotein complexes are structured, comparing different organisms and trying to link these structures to telomere biology. It emerges that telomeres are formed by a complex and specific network of interactions between DNA, RNA and proteins. The fact that these interactions and associated activities are reinforcing each other might help to guaranty the robustness of telomeric functions across the cell cycle and in the event of cellular perturbations. We propose that telomeric nucleoprotein complexes orient cell fate through dynamic transitions in their structures and their organization

Published
2010
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12. Identification and structural basis of the reaction catalyzed by CYP121, an essential cytochrome P450 in Mycobacterium tuberculosis

Author

Mickaël Jacquet, Jean-Baptiste Charbonnier, Olivier Lequin, Robert Thai, Roger Genet, Marie Hélène Le Du, Christophe Dugave, Marie Courçon, Alain Lecoq, Cédric Masson, Alistair J. Fielding, Jean-Luc Pernodet, Muriel Gondry, Pascal Belin, Service d'Ingénierie Moléculaire pour la Santé (ex SIMOPRO) (SIMoS), Médicaments et Technologies pour la Santé (MTS), Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Service de Bioénergétique, Biologie Stucturale, et Mécanismes (SB2SM), Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Synthèse, Structure et Fonction de Molécules Bioactives (SSFMB), Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Service de Chimie Bio-Organique et de Marquage (SCBM), Département d'Ingenierie et d'Etudes des Protéines (DIEP), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institut de génétique et microbiologie [Orsay] (IGM), Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Institut de Biologie et de Technologies de Saclay (IBITECS), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay

Subjects
[SDV.BIO]Life Sciences [q-bio]/Biotechnology, Protein Conformation, Stereochemistry, [SDV]Life Sciences [q-bio], Crystallography, X-Ray, 010402 general chemistry, Peptides, Cyclic, 01 natural sciences, Catalysis, Substrate Specificity, 03 medical and health sciences, chemistry.chemical_compound, Protein structure, Cytochrome P-450 Enzyme System, [SDV.SP.MED]Life Sciences [q-bio]/Pharmaceutical sciences/Medication, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Nuclear Magnetic Resonance, Biomolecular, Heme, Ferredoxin, 030304 developmental biology, 0303 health sciences, Binding Sites, Multidisciplinary, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Ligand, Rational design, Active site, Hydrogen Bonding, Dipeptides, Mycobacterium tuberculosis, Nuclear magnetic resonance spectroscopy, Biological Sciences, 0104 chemical sciences, 3. Good health, Oxygen, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, chemistry, Intramolecular force, biology.protein, [SDV.SP.PHARMA]Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology
Abstract

The gene encoding the cytochrome P450 CYP121 is essential for Mycobacterium tuberculosis . However, the CYP121 catalytic activity remains unknown. Here, we show that the cyclodipeptide cyclo(l-Tyr-l-Tyr) (cYY) binds to CYP121, and is efficiently converted into a single major product in a CYP121 activity assay containing spinach ferredoxin and ferredoxin reductase. NMR spectroscopy analysis of the reaction product shows that CYP121 catalyzes the formation of an intramolecular C-C bond between 2 tyrosyl carbon atoms of cYY resulting in a novel chemical entity. The X-ray structure of cYY-bound CYP121, solved at high resolution (1.4 Å), reveals one cYY molecule with full occupancy in the large active site cavity. One cYY tyrosyl approaches the heme and establishes a specific H-bonding network with Ser-237, Gln-385, Arg-386, and 3 water molecules, including the sixth iron ligand. These observations are consistent with low temperature EPR spectra of cYY-bound CYP121 showing a change in the heme environment with the persistence of the sixth heme iron ligand. As the carbon atoms involved in the final C-C coupling are located 5.4 Å apart according to the CYP121-cYY complex crystal structure, we propose that C-C coupling is concomitant with substrate tyrosyl movements. This study provides insight into the catalytic activity, mechanism, and biological function of CYP121. Also, it provides clues for rational design of putative CYP121 substrate-based antimycobacterial agents.

Published
2009
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13. Structural, thermodynamic, and cellular characterization of human centrin 2 interaction with xeroderma pigmentosum group C protein

Author

Jaime F. Angulo, Yves Blouquit, Thierry Rose, Jean-Baptiste Charbonnier, Simona Miron, Constantin T. Craescu, Emilie Renaud, Patricia Duchambon, Marie Hélène Le Du, Petya Christova, Alexandra Shosheva, Laboratoire de Biologie Structurale et Radiobiologie (LBSR), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Laboratoire de Génétique de la Radiosensibilité (LGR), Imagerie intégrative de la molécule à l'organisme, Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Bulgarian Academy of Sciences (BAS), Biophysique Moléculaire (Plate-forme), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), This work was supported by the Centre National de la Recherche Scientifique, the Institut National de la Santé et de la Recherche Médicale, the Institut Curie, The French/Bulgarian joint programme RILA (no. 09838PF) and Electricité de France (grand no. 8702)., and Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)

Subjects
MESH: Cell Nucleus, Models, Molecular, Cytoplasm, Xeroderma pigmentosum, MESH: Protein Structure, Secondary, Cell Cycle Proteins, Plasma protein binding, MESH: Calcium-Binding Proteins, Biology, Protein Structure, Secondary, Protein–protein interaction, 03 medical and health sciences, MESH: Protein Structure, Tertiary, Protein structure, MESH: Cell Cycle Proteins, Structural Biology, Calcium-binding protein, medicine, MESH: Protein Binding, Humans, Binding site, Molecular Biology, 030304 developmental biology, MESH: Xeroderma Pigmentosum, Cell Nucleus, 0303 health sciences, Xeroderma Pigmentosum, MESH: Humans, Binding Sites, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], MESH: Cytoplasm, 030302 biochemistry & molecular biology, Calcium-Binding Proteins, medicine.disease, Protein Structure, Tertiary, DNA-Binding Proteins, Biochemistry, MESH: Binding Sites, MESH: Calcium, Centrin, Biophysics, Thermodynamics, Calcium, MESH: Thermodynamics, MESH: DNA-Binding Proteins, MESH: Models, Molecular, Nucleotide excision repair, Protein Binding
Abstract

International audience; Human centrin 2 (HsCen2), an EF-hand calcium binding protein, plays a regulatory role in the DNA damage recognition during the first steps of the nucleotide excision repair. This biological action is mediated by the binding to a short fragment (N847-R863) from the C-terminal region of xeroderma pigmentosum group C (XPC) protein. This work presents a detailed structural and energetic characterization of the HsCen2/XPC interaction. Using a truncated form of HsCen2 we obtained a high resolution (1.8 A) X-ray structure of the complex with the peptide N847-R863 from XPC. Structural and thermodynamic analysis of the interface revealed the existence of both electrostatic and apolar inter-molecular interactions, but the binding energy is mainly determined by the burial of apolar bulky side-chains into the hydrophobic pocket of the HsCen2 C-terminal domain. Binding studies with various peptide variants showed that XPC residues W848 and L851 constitute the critical anchoring side-chains. This enabled us to define a minimal centrin binding peptide variant of five residues, which accounts for about 75% of the total free energy of interaction between the two proteins. Immunofluorescence imaging in HeLa cells demonstrated that HsCen2 binding to the integral XPC protein may be observed in living cells, and is determined by the same interface residues identified in the X-ray structure of the complex. Overexpression of XPC perturbs the cellular distribution of HsCen2, by inducing a translocation of centrin molecules from the cytoplasm to the nucleus. The present data confirm that the in vitro structural features of the centrin/XPC peptide complex are highly relevant to the cellular context.

Published
2007
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14. Crystal structure of the complex between the monomeric form of Toxoplasma gondii surface antigen 1 (SAG1) and a monoclonal antibody that mimics the human immune response

Author

Nicole Battail-Poirot, Geneviève Sibaï, Marie Gauthier, Marc Graille, Marie-Hélène Le Du, Odile Letourneur, Frédéric Ducancel, Dominique Rolland, Bruno H. Muller, Enrico A. Stura, Marc Bossus, Département d'Ingenierie et d'Etudes des Protéines (DIEP), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)

Subjects
Protein Conformation, animal diseases, [SDV]Life Sciences [q-bio], Protozoan Proteins, Antibodies, Protozoan, Crystallography, X-Ray, Ligands, Epitope, Epitopes, Mice, 0302 clinical medicine, Protein structure, Structural Biology, conformational epitope, Toxoplasma gondii recombinant SAG1, ComputingMilieux_MISCELLANEOUS, 0303 health sciences, biology, Antibodies, Monoclonal, 3. Good health, Antigens, Surface, Antibody, Crystallization, Toxoplasma, Toxoplasmosis, crystal structure, medicine.drug_class, 030231 tropical medicine, Molecular Sequence Data, Antigens, Protozoan, Monoclonal antibody, 03 medical and health sciences, Antigen, parasitic diseases, medicine, Animals, Humans, Amino Acid Sequence, Molecular Biology, 030304 developmental biology, Binding Sites, Sequence Homology, Amino Acid, Immunodominant Epitopes, Intracellular parasite, Toxoplasma gondii, biology.organism_classification, Virology, Peptide Fragments, biology.protein, Conformational epitope
Abstract

Toxoplasma gondii, the intracellular parasite responsible for toxoplasmosis infects more than one-third of the world population and can be life-threatening for fetuses and immunocompromised patients. The surface protein SAG1 is an important immune target, which provides a strong immune response against the invasive tachyzoite while the other forms of the parasite, devoid of SAG1 at their surface, are multiplying. In addition to this role as a "hot spot" decoy, SAG1 is predicted to act as an adhesin during host-cell attachment through its binding to proteoglycans. To begin to understand the relationships between SAG1 epitopes and the ligand-binding site, we have solved the crystal structure of the monomeric form of T.gondii SAG1 complexed to a Fab derived from a monoclonal antibody raised against tachyzoite particles. This antibody competes strongly with human Toxoplasma-specific sera, suggesting that its epitope is part of an immunodominant region present on the surface of SAG1. The structure reveals that this conformational epitope, located within the SAG1 N-terminal domain, does not overlap with the proposed ligand-binding pocket. This study provides the first structural description of the monomeric form of SAG1, and significant insights into its dual role of adhesin and immune target during parasite infection.

Published
2005
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